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Cited by 225Immune cells in cancerPhagocytosis and Immune RegulationImmune Response and Inflammation

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Characterization of murine macrophages from bone marrow, spleen and peritoneum

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Wang, Changqi, Yu, Xiao, Cao, Qi, Wang, Ya, Zheng, Guoping, Tan, Thian Kui, Zhao, Hong, Zhao, Ye, Wang, Yiping, Harris, David CH·2013·BMC Immunology
DOI 10.1186/1471-2172-14-6PMC PMC3574850
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Abstract

BACKGROUND: Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. However, differences among macrophages from these particular sources remain unclear. In this study, the features of murine macrophages from bone marrow, spleen and peritoneum were compared. RESULTS: We found that peritoneal macrophages (PMs) appear to be more mature than bone marrow derived macrophages (BMs) and splenic macrophages (SPMs) based on their morphology and surface molecular characteristics. BMs showed the strongest capacity for both proliferation and phagocytosis among the three populations of macrophage. Under resting conditions, SPMs maintained high levels of pro-inflammatory cytokines expression (IL-6, IL-12 and TNF-α), whereas BMs produced high levels of suppressive cytokines (IL-10 and TGF-β). However, SPMs activated with LPS not only maintained higher levels of (IL-6, IL-12 and TNF-α) than BMs or PMs, but also maintained higher levels of IL-10 and TGF-β. CONCLUSIONS: Our results show that BMs, SPMs and PMs are distinct populations with different biological functions, providing clues to guide their further experimental or therapeutic use.

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Figures

Figure 1 Morphological characteristics of cultured macrophages derived from spleen (A, D), bone marrow (B, E) and peritoneal cavity (C, F), and their cell size assessment (G). All cells were cultured in complete RPMI1640 on 6-well plates, and after removal of supernatant, cells were then stained with Giemsa-wright dye (A, B, C) and to demonstrate lysosome, anti-LAMP1 (D, E, F) (original magnification x400). Cell size was assessed by flow cytometry analysis (G).
Figure 1 Morphological characteristics of cultured macrophages derived from spleen (A, D), bone marrow (B, E) and peritoneal cavity (C, F), and their cell size assessment (G). All cells were cultured in complete RPMI1640 on 6-well plates, and after removal of supernatant, cells were then stained with Giemsa-wright dye (A, B, C) and to demonstrate lysosome, anti-LAMP1 (D, E, F) (original magnification x400). Cell size was assessed by flow cytometry analysis (G).CC BY · PubMed Central
Figure 2 Expression of surface molecules on resting SPM, BM and PM was determined by flow cytometry. Red solid lines, staining with (A) anti-CD115, anti-CD206, anti-Gr-1, (B) anti-CD80, anti-CD86, anti-MHC II, (C) anti-B7-H1, anti-B7-H2, anti-B7-H3 and anti-B7-H4; grey filled , staining with the relevant isotype controls. The percentage positivity is shown at the upper right of each histogram. Data are representative of 5 separate experiments of each macrophage preparation. D: summary data of surface molecules expression. Data are mean ± SEM. *p < 0.05, **p < 0.01.
Figure 2 Expression of surface molecules on resting SPM, BM and PM was determined by flow cytometry. Red solid lines, staining with (A) anti-CD115, anti-CD206, anti-Gr-1, (B) anti-CD80, anti-CD86, anti-MHC II, (C) anti-B7-H1, anti-B7-H2, anti-B7-H3 and anti-B7-H4; grey filled , staining with the relevant isotype controls. The percentage positivity is shown at the upper right of each histogram. Data are representative of 5 separate experiments of each macrophage preparation. D: summary data of surface molecules expression. Data are mean ± SEM. *p < 0.05, **p < 0.01.CC BY · PubMed Central
Figure 3 Macrophage growth rate treated with different M-CSF concentrations. BM, SPM and PM were cultured with M-CSF in concentrations of 2 ng/ml (A) or 10 ng/ml (B) for 0, 4, 7 and 14 days. The numbers of macrophages were quantified. Images are representative of 3 separate experiments. Data are mean ± SEM. *p < 0.05, **p < 0.01.
Figure 3 Macrophage growth rate treated with different M-CSF concentrations. BM, SPM and PM were cultured with M-CSF in concentrations of 2 ng/ml (A) or 10 ng/ml (B) for 0, 4, 7 and 14 days. The numbers of macrophages were quantified. Images are representative of 3 separate experiments. Data are mean ± SEM. *p < 0.05, **p < 0.01.CC BY · PubMed Central
Figure 4 FITC-dextran uptake assay of macrophages from the three different sources. (A) Purified macrophages were incubated with FITC-dextran at 37°C for 45 min, and then washed extensively to remove excess FITC-dextran, followed by FACS analysis. Representative histograms are shown. Solid grey histograms represent control groups; solid red lines represent the percentage of phagocytic macrophages. (B) Group histograms showing both population and median fluorescence intensity (MFI) values. Data are the mean ± SEM from five separate experiments. *p < 0.05.
Figure 4 FITC-dextran uptake assay of macrophages from the three different sources. (A) Purified macrophages were incubated with FITC-dextran at 37°C for 45 min, and then washed extensively to remove excess FITC-dextran, followed by FACS analysis. Representative histograms are shown. Solid grey histograms represent control groups; solid red lines represent the percentage of phagocytic macrophages. (B) Group histograms showing both population and median fluorescence intensity (MFI) values. Data are the mean ± SEM from five separate experiments. *p < 0.05.CC BY · PubMed Central
Figure 5 Stimulation of CD4+ T cells by macrophages presenting OVA in [3H]-thymidine incorporation assays. Isolated macrophages and dendritic cells (DCs) were loaded with OVA (10 μg/ml) and irradiated; then co-cultured with DO11.10 CD4+ T cells for 48 hours. Cultures were then pulsed with [3H]-thymidine, and incorporated counts determined. DCs were used as positive control. Data are the mean ± SEM from three separate experiments.
Figure 5 Stimulation of CD4+ T cells by macrophages presenting OVA in [3H]-thymidine incorporation assays. Isolated macrophages and dendritic cells (DCs) were loaded with OVA (10 μg/ml) and irradiated; then co-cultured with DO11.10 CD4+ T cells for 48 hours. Cultures were then pulsed with [3H]-thymidine, and incorporated counts determined. DCs were used as positive control. Data are the mean ± SEM from three separate experiments.CC BY · PubMed Central
Figure 6 Cytokine mRNA expression profiles of the three populations (SPMs, BMs and PMs) with and without activation with LPS. mRNA levels of IL-10, TGF-β, IL-6, IL-12 and TNF-α in SPMs, BMs and PMs were measured by real time PCR with β-actin as the housekeeping gene; (n = 5). Values are expressed as 10x (gene of interest vs β-actin). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6 Cytokine mRNA expression profiles of the three populations (SPMs, BMs and PMs) with and without activation with LPS. mRNA levels of IL-10, TGF-β, IL-6, IL-12 and TNF-α in SPMs, BMs and PMs were measured by real time PCR with β-actin as the housekeeping gene; (n = 5). Values are expressed as 10x (gene of interest vs β-actin). *p < 0.05, **p < 0.01, ***p < 0.001.CC BY · PubMed Central

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  1. Cao Q, Zheng D, Sun Y, Wang Y, Lee VW, Zheng G, Alexander SI, Harris DC: Impaired alternative activated macrophages from bone marrow in treatment of CKD: Associations with their proliferation in inflamed kidney. Presented in abstract form at the annual meeting of the American Society of Nephrology; October 27 through November 1. 2009, CA: San Diego

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Metadata

Authors
Wang, C., Yu, X., Cao, Q., Wang, Y., Zheng, G., Tan, T. K., Zhao, H., Zhao, Y., Wang, Y., & Harris, D. C.
Affiliations
  • Westmead Hospital (AU)
  • Shanxi Medical University (CN)
Corresponding author
Wang, Changqi
ORCID
Venue
BMC Immunology
Publisher
Springer Science and Business Media LLC
Published
2013-02-05
Volume
14
Issue
1
Pages
6-6
ISSN
1471-2172 (ISSN-L)
DOI
10.1186/1471-2172-14-6
PMC
PMC3574850
OpenAlex
W2171040124
Cited by
225
References
50
Citations by year
2026: 5 · 2025: 14 · 2024: 18 · 2023: 16 · 2022: 12 · 2021: 13 · 2020: 32 · 2019: 22 · 2018: 25 · 2017: 27 · 2016: 17 · 2015: 17 · 2014: 7
FWCI
7.56
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Immunology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Medical Research Council
  • National Health and Medical Research Council (457345)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Life Sciences › Immunology and Microbiology › Immunology › Phagocytosis and Immune Regulation
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
Keywords
macrophage heterogeneity, peritoneal macrophages, bone marrow-derived macrophages, splenic macrophages, macrophage maturation, phagocytosis, macrophage proliferation, inflammatory cytokines, IL-10, TGF-beta, lipopolysaccharide stimulation, murine macrophages
MeSH terms
Animals, Bone Marrow Cells, Cell Membrane, Cells, Cultured, Dextrans, Flow Cytometry, Gene Expression Regulation, Lymphocyte Activation, Macrophages, Male, Mice, Inbred BALB C, Phagocytosis, Phenotype, RNA, Messenger, Spleen, CD4-Positive T-Lymphocytes, Cytokines, Fluorescein-5-isothiocyanate, Macrophages, Peritoneal, Antigen Presentation, Cell Shape, Cell Proliferation, Mice

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